A project beginning last year and continuing to date, was started by postdoctoral fellow Leticia Cano. It attempts to identify biomarkers in autoimmune disease using protein profiling in combination with mass spectrometry. Proteins isolated from tissue and immune complexes are used as a source of biomarkers. Collaborations have been established with several clinical groups involved in Sjogren's disease, systemic lupus erythematosus and inflammatory myositis (G. Illei (NICDR), F. Miller (NEIHS), E. Rushing (AFIP), Anil Chauhan (Progen Biologics), Craig Blackstone (NINDS), and Howard Jaffe (NINDS). We have also received OHSR approval to obtain human samples from the Cooperative Human Tissue Network (CHTN) and AFIP (Rushing). Tissues obtained from healthy controls and disease controls have been examined to ensure that we are not merely identifying markers of inflammation. New ways to separate and profile proteins prior to LC/MS/MS analysis are also being investigated, i.e., chromatofocusing, ion exchange, salt precipitations and affinity columns. As an example of the latter, we have isolated circulating immune complexes (CICs) from the plasma of patients with dermatomyositis (DM) and systemic lupus erythematosus (SLE) using a receptor-based affinity column from ProGen Biologics (Chauhan). We have found many proteins that were previously identified as candidate biomarkers for autoimmune diseases. The presence of these proteins in circulating immune complexes suggests that they are targeted by the immune system and that autoantibody assays for these proteins may be developed as biomarkers for systemic autoimmune diseases. We have begun using ELISAs to verify these findings and to show the utility of this approach in defining new biomarkers for immune-mediated diseases. A Beckman PF2D system for 2D-HPLC separation of intact proteins has been acquired and will be used for examining the spastic paraplegia samples (Blackstone). In a recent collaborations we identified a 225kDa marker belonging to myosin heavy chain IIA (MYHIIA) that reacts specifically with chronic lymphocytic leukemia antibody. The MYHIIA form structures on the surface of the HEp-2 cells which are undergoing stress. This suggests that chronic lymphocytic leukemia antibody may bind to surface markers that are the consequence of stress related cell events.